Stats are transcription factors that integrate cytokine and growth factor receptor signaling and are required for normal cell growth, survival, differentiation, and motility. Stat activation is commonly seen in cancer and in particular activated Stat3 is found in ~50% of breast cancer-derived cell lines and in primary breast adenocarcinomas and likely correlates with poor prognosis. The evidence that activated Stat3 contributes mechanistically to breast tumorigenesis is based on cell line studies that have shown that enforced expression of a constitutively activated form of Stat3 (Stat3C) promotes invasiveness and tumorigenicity. Conversely, inhibition of Stat3 activity/expression in cell lines expressing high levels of Stat3 reduces invasiveness, tumorigenicity and angiogenesis. Our studies reveal a prominent role for IL-6 in driving Stat3 activation in breast cancer cell lines through the gp130/Jak pathway providing evidence for an IL-6/Stat3 autocrine/paracrine positive feedback loop in tumorigenesis. However, these cell line based assays cannot adequately address many critical aspects of de novo tumorigenesis including initiation, progression, and metastatic spread which involve tumor:host interactions best evaluated in animal model systems. We have developed the first in vivo model systems in which activated Stat3 can be conditionally regulated in the mammary gland. Preliminary data demonstrates enforced expression of Stat3C in the virgin gland mediates production of pro-inflammatory cytokines and recruitment of leukocytes. We hypothesize that in breast cancer, sustained activation of epithelial cell Stat3, will trigger chronic inflammatory changes, i.e., "wounds that never heal" and thereby promote de novo tumorogenesis(Aim1). In cooperation with established murine mammary tumor models, preliminary data suggests that inducible expression of Stat3C potentiates hyperplasia, increases tumor multiplicity and enhances metastatic disease. We will determine the cooperative effects of Stat3C on tumor latency, histologic progression, leukocyte involvement and metastatic progression. (Aim 2). In addition, we have conditionally deleted Stat3 from the mammary gland in order to examine the functional requirement of Stat3 activation on MMTV-oncogene mediated tumorigenesis as well as human breast cancer derived cell lines. Our preliminary data suggest a requirement for Stat3 in mediating both the growth and metastatic potential of these tumors. These findings complement those in Aims1&2 and combined will allow us to elucidate the Stat3 regulated contributions to the promotion of murine and human mammary cell tumorigenesis (Aim3). IL-6 is a principal mediator of Stat3 activation in primary breast tumors as well as breast cancer derived cell lines. We hypothesize that Stat3 becomes activated by IL-6 and that Stat3 in conjunction with NF-kB potentiates IL-6 production establishing a positive feed-back loop in cancer. We will identify the precise contribution of NF-kB and Stat3 to IL-6 regulation in breast cancer derived cell lines. We will quantify the levels of and distribution of pStat3, IL-6 and NF-kB and leukocyte infiltration in archived breast cancer samples with matched lymph nodes and distant metastasis. We hypothesize that the IL-6/Stat3/NF-kB trio will be predictive of metastatic progression (Aim 4). Using these model systems, which can alternatively turn Stat3 activation on and off, we are now poised to fully investigate the biological role of Stat3 in breast tumorigenesis in vivo. The results of this work will demonstrate the functional consequences of this activated transcription factor to de novo tumorigenesis, tumor progression and metastatic disease and establish the rationale for targeted therapies aimed at the Jak/Stat3 pathway in breast cancer. [unreadable] PUBLIC HEALTH RELEVANCE: Breast cancer is the most common malignancy diagnosed among women worldwide. Despite significant improvements in the diagnosis and treatment of this disease, tumor dormancy followed by distant recurrences accounts for 90% of all cancer deaths. This suggests that malignant cells have already disseminated at the time of diagnosis. Micrometastasis in the blood and bone marrow are the principal targets for adjuvant chemotherapy and hormonal therapy. However, these metastatic cells frequently evade therapeutic interventions and eventually recur. Clearly understanding the molecular mechanisms underlying the development of metastatic disease is required in order to treat this fatal disorder effectively. The development of animal models which recapitulate human breast tumorigenesis is instrumental to our understanding of this complex disease. Such models are essential for the rational development and testing of targeted therapeutics. We have determined that a normal cellular protein, Stat3, is persistently activated or "turned-on" in many cancers including >30% of breast cancers. Stat3 is activated in breast cancers by a group of proteins known as inflammatory cytokines which are important mediators of wound healing but can also potentiate the metastatic spread of cancer cells. Inactivating or "removing" activated Stat3 from selected breast cancer cells can alter their ability to migrate and recruit the necessary blood supply for growth and spread. By genetically changing a normal Stat3 into one that is constitutively active, one can make a normal breast cell behave as a cancerous one. We have developed an animal model in which we can "turn-on" or "turn-off" activated Stat3 in the breast gland. Our preliminary data suggests that "turning-on" activated Stat3 may accelerate the development of metastatic disease, while "turning Stat3 off" inhibits tumor growth and metastatic spread. The results of the proposed work will demonstrate the functional consequences of activated Stat3 to breast tumor formation, progression and metastatic disease and establish the rationale for targeted therapies aimed at the Stat3 pathway in breast cancer. [unreadable] [unreadable] [unreadable]